Recent advances in the pharmaceutical industry have resulted in the formation of an increasing number of potential therapeutic agents. However, formulating the compounds for effective oral bioavailability has proven difficult because of problems associated with uptake and high susceptibility to metabolic enzymes.
Natural transporter proteins are involved in the uptake of various molecules into and/or through cells. In general, two major transport systems exist: solute carrier-mediated systems and receptor mediated systems. Carrier-mediated systems use transport proteins that are anchored to the cell membrane, typically by a plurality of membrane-spanning loops and function by transporting their substrates via an energy-dependent flip-flop or other mechanism, exchange and other facilitative or equilibrative mechanisms. Carrier-mediated transport systems are involved in the active or non-active, facilitated transport of many important nutrients such as vitamins, sugars, and amino acids. The carrier systems result in transport into the enterocytes from blood or lumen, and across the epithelial cell layer from lumen into blood (absorption) or blood to lumen (secretion). Carrier-mediated transporters are also present in organs such as the liver and kidney, in which the proteins are involved in the excretion or re-absorption of circulating compounds.
Receptor-mediated transport systems differ from the carrier-mediated systems in that these systems usually utilize proteins that span the cell membrane only a single time. Furthermore, substrate binding triggers an invagination and encapsulation process that results in the formation of various transport vesicles to carry the substrate (and sometimes other molecules) into and through the cell. This process of membrane deformations that result in the internalization of certain substrates and their subsequent targeting to certain locations in the cytoplasm is generally referred to as endocytosis.
Polar or hydrophilic compounds are typically poorly absorbed through an animal's intestine as there is a substantial energetic penalty for passage of such compounds across the lipid bilayers that constitute cellular membranes. Many nutrients that result from the digestion of ingested foodstuffs in animals, such as amino acids, di- and tripeptides, monosaccharides, nucleosides and water-soluble vitamins, are polar compounds whose uptake is essential to the viability of the animal. For these substances there exist specific mechanisms for active transport of the solute molecules across the intestinal epithelia. This transport is frequently energized by co-transport of ions down a concentration gradient.
Known examples of solute carrier systems include two peptide transporters, PEPT1 and PEPT2. The endogenous substrates for these transporters are small peptides consisting of two or three amino acids. These transporters function in the absorption of peptides arising from the digestion of dietary proteins (small intestine) and in the reabsorption of peptides present in the glomerular filtrate.
The human intestinal peptide transporter (PEPT1) and the human kidney peptide transporter (PEPT2) exhibit about 48% identity at the amino acid level. Neither peptide transporter shows significant sequence identity to other known mammalian sequences—they are both about 20% identical to PHT1 and PHT2. The two transporters show some differences in the recognition of β-lactam antibiotics as substrates as well as their marked differences in affinity for many substrates. As such, PEPT1 is a high capacity, low-affinity transporter and PEPT2 is a high affinity transporter. Both transporters accept small peptides as substrates and are driven by a transmembrane electrochemical H+ gradient.
PEPT1 and PEPT2 have been reported to show different patterns of expression in different human tissues. PEPT1 has been reported to be expressed predominantly in the intestine, and also in the kidney (pars convoluta), and liver, with small amounts of expression in the brain and pancreas. Fei et al., Nature 386:563-566 (1994) and Miyamoto et al., Biochimica et Biophysica Acta 1305:34-38 (1996). By contrast, PEPT2 has been reported to be expressed in the kidney and brain, with lower expression reported in the lung, liver and heart and no expression reported in the small intestine. Liu et al., Biochimica et Biophysica Acta 1235:461-466 (1995) and Boll et al., Proc. Natl. Acad. Sci. USA 93:284-289 (1996) and Saito et al., Biochimica et Biophysica Acta 1280:173-177 (1996). Because of the view that PEPT1 and not PEPT2 is expressed in the intestine, existing efforts to improve oral delivery of drugs via peptide transporters have focused on identifying pharmacological agents that are, or can be modified to be, substrates for PEPT1.